Reinier A. Boon

MicroRNA-34a regulates cardiac ageing and function

Ageing is the predominant risk factor for cardiovascular diseases and contributes to a significantly worse outcome in patients with acute myocardial infarction. MicroRNAs (miRNAs) have emerged as crucial regulators of cardiovascular function and some miRNAs have key roles in ageing. We propose that altered expression of miRNAs in the heart during ageing contributes to the age-dependent decline in cardiac function. Here we show that miR-34a is induced in the ageing heart and that in vivo silencing or genetic deletion of miR-34a reduces age-associated cardiomyocyte cell death. Moreover, miR-34a inhibition reduces cell death and fibrosis following acute myocardial infarction and improves recovery of myocardial function. Mechanistically, we identified PNUTS (also known as PPP1R10) as a novel direct miR-34a target, which reduces telomere shortening, DNA damage responses and cardiomyocyte apoptosis, and improves functional recovery after acute myocardial infarction. Together, these results identify age-induced expression of miR-34a and inhibition of its target PNUTS as a key mechanism that regulates cardiac contractile function during ageing and after acute myocardial infarction, by inducing DNA damage responses and telomere attrition.

Long Noncoding RNA MALAT1 Regulates Endothelial Cell Function and Vessel Growth

Rationale: The human genome harbors a large number of sequences encoding for RNAs that are not translated but control cellular functions by distinct mechanisms. The expression and function of the longer transcripts namely the long noncoding RNAs in the vasculature are largely unknown. Objective: Here, we characterized the expression of long noncoding RNAs in human endothelial cells and elucidated the function of the highly expressed metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). Methods and Results: Endothelial cells of different origin express relative high levels of the conserved long noncoding RNAs MALAT1, taurine upregulated gene 1 (TUG1), maternally expressed 3 (MEG3), linc00657, and linc00493. MALAT1 was significantly increased by hypoxia and controls a phenotypic switch in endothelial cells. Silencing of MALAT1 by small interfering RNAs or GapmeRs induced a promigratory response and increased basal sprouting and migration, whereas proliferation of endothelial cells was inhibited. When angiogenesis was further stimulated by vascular endothelial growth factor, MALAT1 small interfering RNAs induced discontinuous sprouts indicative of defective proliferation of stalk cells. In vivo studies confirmed that genetic ablation of MALAT1 inhibited proliferation of endothelial cells and reduced neonatal retina vascularization. Pharmacological inhibition of MALAT1 by GapmeRs reduced blood flow recovery and capillary density after hindlimb ischemia. Gene expression profiling followed by confirmatory quantitative reverse transcriptase-polymerase chain reaction demonstrated that silencing of MALAT1 impaired the expression of various cell cycle regulators. Conclusions: Silencing of MALAT1 tips the balance from a proliferative to a migratory endothelial cell phenotype in vitro, and its genetic deletion or pharmacological inhibition reduces vascular growth in vivo.

MicroRNA-29 in Aortic Dilation: Implications for Aneurysm Formation

Rationale: Aging represents a major risk factor for coronary artery disease and aortic aneurysm formation. MicroRNAs (miRs) have emerged as key regulators of biological processes, but their role in age-associated vascular pathologies is unknown. Objective: We aim to identify miRs in the vasculature that are regulated by age and play a role in age-induced vascular pathologies. Methods and Results: Expression profiling of aortic tissue of young versus old mice identified several age-associated miRs. Among the significantly regulated miRs, the increased expression of miR-29 family members was associated with a profound downregulation of numerous extracellular matrix (ECM) components in aortas of aged mice, suggesting that this miR family contributes to ECM loss, thereby sensitizing the aorta for aneurysm formation. Indeed, miR-29 expression was significantly induced in 2 experimental models for aortic dilation: angiotensin II-treated aged mice and genetically induced aneurysms in Fibulin-4R/R mice. More importantly, miR-29b levels were profoundly increased in biopsies of human thoracic aneurysms, obtained from patients with either bicuspid (n=79) or tricuspid aortic valves (n=30). Finally, LNA-modified antisense oligonucleotide-mediated silencing of miR-29 induced ECM expression and inhibited angiotensin II-induced dilation of the aorta in mice. Conclusion: In conclusion, miR-29-mediated downregulation of ECM proteins may sensitize the aorta to the formation of aneurysms in advanced age. Inhibition of miR-29 in vivo abrogates aortic dilation in mice, suggesting that miR-29 may represent a novel molecular target to augment matrix synthesis and maintain vascular wall structural integrity.

MicroRNA-29 in aortic dilation: implications for aneurysm formation [Molecular Medicine]

Rationale: Aging represents a major risk factor for coronary artery disease and aortic aneurysm formation. MicroRNAs (miRs) have emerged as key regulators of biological processes, but their role in age-associated vascular pathologies is unknown. Objective: We aim to identify miRs in the vasculature that are regulated by age and play a role in age-induced vascular pathologies. Methods and Results: Expression profiling of aortic tissue of young versus old mice identified several age-associated miRs. Among the significantly regulated miRs, the increased expression of miR-29 family members was associated with a profound downregulation of numerous extracellular matrix (ECM) components in aortas of aged mice, suggesting that this miR family contributes to ECM loss, thereby sensitizing the aorta for aneurysm formation. Indeed, miR-29 expression was significantly induced in 2 experimental models for aortic dilation: angiotensin II-treated aged mice and genetically induced aneurysms in Fibulin-4R/R mice. More importantly, miR-29b levels were profoundly increased in biopsies of human thoracic aneurysms, obtained from patients with either bicuspid (n=79) or tricuspid aortic valves (n=30). Finally, LNA-modified antisense oligonucleotide-mediated silencing of miR-29 induced ECM expression and inhibited angiotensin II-induced dilation of the aorta in mice. Conclusion: In conclusion, miR-29-mediated downregulation of ECM proteins may sensitize the aorta to the formation of aneurysms in advanced age. Inhibition of miR-29 in vivo abrogates aortic dilation in mice, suggesting that miR-29 may represent a novel molecular target to augment matrix synthesis and maintain vascular wall structural integrity.

MicroRNAs in myocardial infarction

MicroRNAs (miRNAs) are small noncoding RNAs that block translation or induce degradation of mRNA and thereby control patterns of gene expression. Acute myocardial infarction is a common cardiovascular event that results in cardiac remodelling and can consequently lead to the development of chronic heart failure. Several miRNAs have been shown to control important processes that contribute to the pathophysiological consequences of acute myocardial infarction. miRNAs can either promote or inhibit cardiomyocyte cell death, and also regulate postischaemic neovascularization. Cardiac regeneration can also be regulated by miRNAs that control cardiomyocyte proliferation or interfere with cardioprotective effects mediated by stem or progenitor cells. miRNAs can also be used for direct reprogramming of cardiac fibroblasts into cardiomyocytes. In this Review, we focus on the current understanding of the role of miRNAs in these processes, and particularly discuss the therapeutic potential of miRNAs in treating acute myocardial infarction.

KLF2 Primes the Antioxidant Transcription Factor Nrf2 for Activation in Endothelial Cells

Objective—Atheroprotective blood flow induces expression of anti-inflammatory Krüppel-like factor 2 (KLF2) and activates antioxidant transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) in vascular endothelium. Previously, we obtained KLF2-induced gene expression profiles in ECs, containing several Nrf2 target genes. Our aim was to investigate the role of KLF2 in shear stress–mediated activation of Nrf2 in human umbilical vein endothelial cells (HUVECs). Methods and Results—Expression of Nrf2 and its targets NAD(P)H dehydrogenase quinone 1 (NQO1) and heme oxygenase (HO-1) was elevated by shear and KLF2. KLF2 knockdown showed that shear-induced expression of NQO1 but not Nrf2 was dependent on KLF2. KLF2 overexpression in absence of flow resulted in more efficient activation of Nrf2 by tert-butyl hydroquinone (tBHQ) through enhanced nuclear localization, and promoted expression of a large panel of Nrf2-dependent genes resulting in superior protection against oxidative stress. Comparison of shear-, KLF2-, and Nrf2-induced transcriptomes showed that the majority of shear-modulated gene sets is influenced by KLF2 or Nrf2. Conclusions—We report that KLF2 substantially enhances antioxidant activity of Nrf2 by increasing its nuclear localization and activation. The synergistic activity of these two transcription factors forms a major contribution to the shear stress–elicited transcriptome in endothelial cells.

Class IIb HDAC6 regulates endothelial cell migration and angiogenesis by deacetylation of cortactin

Histone deacetylases (HDACs) deacetylate histones and non‐histone proteins, thereby affecting protein activity and gene expression. The regulation and function of the cytoplasmic class IIb HDAC6 in endothelial cells (ECs) is largely unexplored. Here, we demonstrate that HDAC6 is upregulated by hypoxia and is essential for angiogenesis. Silencing of HDAC6 in ECs decreases sprouting and migration in vitro and formation of functional vascular networks in matrigel plugs in vivo. HDAC6 regulates zebrafish vessel formation, and HDAC6‐deficient mice showed a reduced formation of perfused vessels in matrigel plugs. Consistently, overexpression of wild‐type HDAC6 increases sprouting from spheroids. HDAC6 function requires the catalytic activity but is independent of ubiquitin binding and deacetylation of α‐tubulin. Instead, we found that HDAC6 interacts with and deacetylates the actin‐remodelling protein cortactin in ECs, which is essential for zebrafish vessel formation and which mediates the angiogenic effect of HDAC6. In summary, we show that HDAC6 is necessary for angiogenesis in vivo and in vitro, involving the interaction and deacetylation of cortactin that regulates EC migration and sprouting.

Identification and Characterization of Hypoxia-Regulated Endothelial Circular RNA

RATIONALE Circular RNAs (circRNAs) are noncoding RNAs generated by back splicing. Back splicing has been considered a rare event, but recent studies suggest that circRNAs are widely expressed. However, the expression, regulation, and function of circRNAs in vascular cells is still unknown. OBJECTIVE Here, we characterize the expression, regulation, and function of circRNAs in endothelial cells. METHODS AND RESULTS Endothelial circRNAs were identified by computational analysis of ribo-minus RNA generated from human umbilical venous endothelial cells cultured under normoxic or hypoxic conditions. Selected circRNAs were biochemically characterized, and we found that the majority of them lacks polyadenylation, is resistant to RNase R digestion and localized to the cytoplasm. We further validated the hypoxia-induced circRNAs cZNF292, cAFF1, and cDENND4C, as well as the downregulated cTHSD1 by reverse transcription polymerase chain reaction in cultured endothelial cells. Cloning of cZNF292 validated the predicted back splicing of exon 4 to a new alternative exon 1A. Silencing of cZNF292 inhibited cZNF292 expression and reduced tube formation and spheroid sprouting of endothelial cells in vitro. The expression of pre-mRNA or mRNA of the host gene was not affected by silencing of cZNF292. No validated microRNA-binding sites for cZNF292 were detected in Argonaute high-throughput sequencing of RNA isolated by cross-linking and immunoprecipitation data sets, suggesting that cZNF292 does not act as a microRNA sponge. CONCLUSIONS We show that the majority of the selected endothelial circRNAs fulfill all criteria of bona fide circRNAs. The circRNA cZNF292 exhibits proangiogenic activities in vitro. These data suggest that endothelial circRNAs are regulated by hypoxia and have biological functions.

Reduced MicroRNA-150 Is Associated with Poor Survival in Pulmonary Arterial Hypertension

RATIONALE MicroRNAs (miRNAs or miRs) are implicated in the pathogenesis of various cardiovascular diseases, including pulmonary arterial hypertension (PAH). OBJECTIVES We sought to measure changes in plasma levels of miRNAs in patients with PAH and relate them to the severity of the disease. METHODS A microarray screen was performed on total plasma RNA from eight patients with PAH and eight healthy control subjects. Quantitative polymerase chain reaction confirmed reduced miR-150 concentrations and was then used to measure miR-150 levels in (1) two separate cohorts of patients with PAH, from London (n = 145) and Sheffield (n = 30), respectively; (2) circulating microvesicles and blood cells; and (3) lungs from a monocrotaline rat model. MEASUREMENTS AND MAIN RESULTS Fifty-eight miRNAs showed differences in plasma concentration and miR-150 the largest down-regulation in PAH. Receiver-operator-characteristic analysis showed both raw and normalized plasma miR-150 levels correlated with 2-year survival (P < 0.01) in patients with PAH. Cox regression analysis confirmed miR-150 levels as a significant predictor of survival. Age, baseline cardiac index, World Health Organization functional class, 6-minute walk distance, disease duration, and red cell distribution width also predicted survival. Entering these covariates in a multivariable model verified plasma miR-150 levels as an independent predictor of survival in PAH (hazard ratio, 0.533; P = 0.010). miR-150 levels also predicted survival in a second, independent PAH cohort. miR-150 levels were significantly reduced in circulating microvesicles from patients with PAH and the lungs of the monocrotaline rat. CONCLUSIONS Reduced circulating miR-150 levels are associated with poor survival in PAH.

Long Noncoding RNAs: From Clinical Genetics to Therapeutic Targets?

Recent studies suggest that the majority of the human genome is transcribed, but only about 2% accounts for protein-coding exons. Long noncoding RNAs (lncRNAs) constitute a heterogenic class of RNAs that includes, for example, intergenic lncRNAs, antisense transcripts, and enhancer RNAs. Moreover, alternative splicing can lead to the formation of circular RNAs. In support of putative functions, GWAS for cardiovascular diseases have shown predictive single-nucleotide polymorphisms in lncRNAs, such as the 9p21 susceptibility locus that encodes the lncRNA antisense noncoding RNA in the INK4 locus (ANRIL). Many lncRNAs are regulated during disease. For example, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and myocardial infarction-associated transcript (MIAT) were shown to affect endothelial cell functions and diabetic retinopathy, whereas lincRNA-p21 controls neointima formation. In the heart, several lncRNAs were shown to act as microRNA sponges and to control ischemia-reperfusion injury or act as epigenetic regulators. In this review, the authors summarize the current understanding of lncRNA functions and their role as biomarkers in cardiovascular diseases.